Non-toxic immune stimulating enterotoxin compositions

ABSTRACT

Pyrogenic toxins, such as  staphylococcal  enterotoxins, modified in the disulfide loop region are provided. The modified toxins retain useful biological properties but have substantially reduced toxicity compared to the corresponding unmodified native toxin. The native pyrogenic toxins are typically modified by deletions within the disulfide loop region to produce modified enterotoxins having 100-fold or greater decrease in toxicity.

This application is a national phase filing of International PatentApplication PCT/US98/25107 filed Dec. 1, 1998, which claims benefit ofU.S. Provisional Application No. 60/067,357 filed on Dec. 2, 1997.

GOVERNMENT SUPPORT

This invention was funded in part by the United States Department ofHealth under NIH grant R11A128401. The United States Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

A group of biological agents termed superantigens has been describedbased on their ability to stimulate monocytes and unprimed CD4⁺ (MHCClass II restricted) and CD8⁺ (MHC Class I restricted) T-cells. Includedwith the designation of superantigens are the enterotoxins ofStaphylococcus aureus.

The enterotoxins of Staphylococcus aureus form a group of serologicallydistinct proteins, originally designated A, B, C₁, C₂, C₃, D, E, G, andH. Subsequently, a number of variants have been described. Theseproteins and toxic shock syndrome proteins were originally recognized asthe causative agents of staphylococcal food poisoning. Ingestion ofpreformed enterotoxin in contaminated food leads to the rapiddevelopment (within two to six hours) of symptoms of vomiting anddiarrhea that are characteristic of staphylococcal food poisoning. Toxicshock syndrome toxin-1, TSST- 1, a distantly related protein alsoproduced by S. aureus, is classically responsible for the toxic shocksyndrome, although other staphylococcal enterotoxins may result in thesyndrome due to the induction of cytokines.

Other agents which have been identified as superantigens include forexample, the staphylococcal exfoliative toxins, A and B; the mammarytumor virus superantigen; rabies virus nucleocapsid protein; pyrogenicexotoxins A, B, C from S. pyrogens; and the Mycoplasma arthritidesmitogen. Additional biological agents which have been demonstrated tohave superantigen properties include the human immunodeficiency virus(HIV), gp120 and peptides from HIV, mouse mammary tumor virus and felineimmunodeficiency virus. A Leishmania peptide antigen has also beendisclosed as a superantigen.

Superantigens, unlike conventional antigens, do not require processingin-vivo. In general, superantigens have two binding regions, one ofwhich interacts with the Class II major histocompatibility complex (MHC)on the antigen presenting cell and the other which interacts with the Vβvariable region of the T-cell receptor on CD4 and/or CD8 cells. Variousenterotoxins bind to one or more of the different Vβ receptor epitopes.In contrast to conventional antigens, superantigens do not occupy theT-cells receptor cleft but are felt to bind to an external region thusexplaining the ability to activate a broad population of T-cells.

Enterotoxins produced by Staphylococcus aureus include a group ofrelated proteins of about 20 to 30 Kd. The complete amino acidcomposition of a number of staphylococcal enterotoxins and streptococcalpyrogenic exotoxin has been reported (see e.g., PCT Patent Appl. No. WO93/24136.)

Staphylococcal enterotoxins (“SEs”) were initially classified on thebasis of their antigenic properties into groups A, B, C1, C2, C3, D, andE. Subsequent relatedness was based on peptide and DNA sequence data.Among the staphylococcal enterotoxins, groups B and C are closelyrelated and groups A, D, and E are closely related in amino acidsequence. SEC1, SEC2, and SEC3 and related isolates share approximately95% sequence similarity. Table 1 shows the alignment of the predictedsequences of the eight known SEC variants following cleavage of thesignal peptide. The N-terminus of each of the mature proteins wasverified by amino acid sequencing. Amino acid positions that containresidues that are not conserved among all SEC are indicated byasterisks. SEB and SEC are approximately 45-50% homologous. In contrast,non-enterotoxin superantigens, TSST-1 and Streptococcal PyrogenicEnterotoxin C (SPEC) share only approximately 20% primary sequencehomology to SEC. Despite these differences, the tertiary structure ofthe various enterotoxins show nearly identical folds.

The staphylococcal enterotoxins A, B, C₁, C₂, C₃, D, E, G and H share acommon structural feature of a disulfide bond not present in otherenterotoxins. Table 2 shows the position of the disulfide bond in anumber of enterotoxins. Data in reference to the active sites of theenterotoxin molecule in relationship to biological activity, MHCbinding, and TCR binding has been obtained. Sequence data demonstrate ahigh degree of similarity in four regions of the enterotoxins (See Table3). The peptides implicated in potential receptor binding correspond toregions 1 and 3 which form a groove in the molecule. Amino acid residueswithin and adjacent to the α₃ cavity of SEC3 have been shown to relateto T-cell activation.

TABLE 2 LOCATION OF DISULFIDE LOOP IN STAPHYLOCOCCUS ENTEROTOXINS AMINOACID ENTEROTOXIN RESIDUES AMINO ACID SEQUENCE OF DISULFIDE LOOP SEA96-106 96?CAGGTPNKTAC (SEQ. ID. NO:9) SEB 93-11493?CYFSKKTNDINSHQTPKRKTC (SEQ. ID. NO:10) SEC1 93-11093?CYFSSKDNVGKVTGGKTC (SEQ. ID. NO:11) SEC2 93-110 93?CYFSSKDNVGKVTGGKTC(SEQ. ID. NO:12) SEC3 FRI 913 93-110 93?CYFSSKDNVGKVTGGKTC (SEQ. ID.NO:13) SEC3 FRI 909 93-110 93?CYFSSKDNVGKVTSGKTC (SEQ. ID. NO:14) SEC4446 93-110 93?CYFSSKDNVGKVTGGKTC (SEQ. ID. NO:15) SEC-Bovine 93-11093?CYFSSKDNVGKVTGGKTC (SEQ. ID. NO:16) SEC-Ovine 93-11093?CCFSSKDNVGKVTGGKTC (SEQ. ID. NO:17)

The staphylococcal enterotoxins are potent activators of T-cells,resulting in proliferation and the generation of cytotoxic T-cells. SEAis a potent T-cell mitogen eliciting strong polyclonal activation atconcentrations of 10⁻¹³ to 10⁻¹⁰ molar in human systems.

The staphylococcal enterotoxins, aside from the acute gastroenteritisand toxic shock syndrome associated with them, have been shown to have avariety of other beneficial biological effects. The biological effectsof these agents and the toxic shock syndrome are due in part to theability of staphylococcal enterotoxins to induce cytokines. Variouscytokines described include IL-1, IL-2, and tumor necrosis factor(“TNF”). More recently SEB and toxic shock syndrome toxin (“TSST-1”)have been shown to induce interleukin-12, an inducer of cell mediatedimmunity, in human peripheral blood mononuclear cells. (See Leung etal., J Exp Med, 181:747 (1995)). The antitumor activity of treatingcancer in rabbits utilizing 40 to 60 μg/kg of a staphylococcalenterotoxin has been disclosed in PCT Patent Appl. Nos. WO 91/10680 andWO 93/24136.

Exposure to enterotoxin either in-vitro or in-vivo leads to depletion ofT-cells having the appropriate Vβ receptor through programmed cell deathin some strains of mice, specifically Balb/c and CBA/2. Cell death canbe prevented by high doses of retinol or RU-38486. Programmed cell deathhas not been observed upon exposure of human cells to enterotoxins.

Although the systemic lethal toxicity of enterotoxins has been relatedto their ability to induce cytokines, particularly IL-1, IL-2 and gammainterferon, lethal toxicity also appears to be related to a synergisticactivity with endogenous endotoxins and the ability of the liver todetoxify endotoxins. Although a number of animals have been utilized toevaluate lethality, the accepted model is the continous infusion over aperiod of time, usually 4 days, in rabbits. The direct toxic dose variesamong various species. The 50% lethal dose of TSST-1 is approximately 50μg/kg for Balb/c mice. Piglets, although showing clinical manifestationsof toxic shock syndrome, tolerate doses of 100 μg/kg of TSST-1.TSST-ovine is known to be non-toxic at doses of 200 μg in rabbits.

In Dutch belted rabbits, intramuscular injection of 50 mg/kg ofstaphylococcal enterotoxin B caused death. Intravenous injection at 0.5to 1.0 mg/kg of enterotoxin A or B in rhesus macaques results inhypotension and death (Liu C. T., et al Amer J Vet Res 39:279 and 1213,1978).

In contrast to other species, man is extremely sensitive toenterotoxins. One (1) mg of TSST-1, approximately 15 nanogram/kg, can belethal for man. Therefore, the recommended doses currently proposed inthe art for treating man are unacceptable. There is a need, therefore,for mutant staphylococcal enterotoxins which are non-toxic atanticipated doses for man while still retaining desirable biologicalactivity.

Several studies of staphylococcal enterotoxin have identified a numberof biologically active modified or mutant enterotoxins with reducedtoxicity. Carboxymethylation of SEB results in a loss ofgastrointestinal toxicity but not mitogenic activity. Studies with theTSST-1 have demonstrated the active site to be between amino acidsresidue 115 and 141. Point mutation of site 135 from histidine toalanine results in a loss of mitogenic activity and toxicity (SeeBonventre P. F., et al. Infect Immun 63:509(1995)). Studies with thestaphylococcal enterotoxin SEC1 demonstrated that the disulfide bondbetween residue 93 and 110 is not required for activity (See Hovde etal., Mol Microbiol 13:897 (1994)). Studies of the molecular bindingregion of staphylococcal enterotoxin B using overlapping peptidesdemonstrated peptide 124 to 154 inhibited SEB induced mitogenicactivity.

Based on the known biological activities of the toxic nativeenterotoxins, it is desirable to create mutants which are at least1000-fold or more less toxic compared to native enterotoxins and retainbiological activity. Recent studies have demonstrated that mutantenterotoxins can be produced which retain certain biological activitiesand which may be significantly less lethal as determined in rabbits. Amutant of the TSST-1 enterotoxin which differs in amino acid 136 and isnon-lethal at ten times the lethal dose of the native toxin (inrabbits), but retains biological activity has been disclosed. A numberof mutants of SEC1, unable to form a disulfide bond, have been reportedto be ten times less toxic than the native toxin while retainingbiological activity. (See e.g., Hovde et al., Molec Microbiol 13:897(1994)).

SUMMARY OF THE INVENTION

The present invention relates to modified versions of disulfideloop-containing bacterial pyrogenic toxins. The modified pyrogenictoxins retain useful biological properties but have substantiallyreduced toxicity (e.g., toxicity reduced by at least about 10-fold)compared to the corresponding unmodified native toxin. Selecteddeletions within the disulfide loop region can produce modified toxinshaving a 100-fold or greater decrease in toxicity. The toxicity of themodified toxin can be measured based on a variety of parameters,including emetic response inducing activity, fever inducing activity,and lethality (as measured by LD₅₀ in Dutch Belted rabbits).

Examples of the present modified toxins include disulfide loop regiondeletion mutants of native toxins derived from Staphylococcus aureus orStreptococcus pyrogenes. Suitable native disulfide loop-containingtoxins which may be modified according to the present invention includeType A, B, C, D and E staphylococcal enterotoxins as well asstreptococcal pyrogenic enterotoxin A (“SPEA”) and streptococcalsuperantigen (“SSA”) produced by S. pyrogenes.

The pyrogenic toxins constitute a family of exotoxins produced byspecies of gram positive cocci, such as Staphylococcus andStreptococcus. The pyrogenic toxins are characterized by shared abilityto induce fever, enhance host susceptibility to endotoxin shock, andinduce T cell proliferation through action as superantigens. Examples ofpyrogenic toxins include TSST-1, staphylococcal enterotoxins (SEs), andstreptococcal pyrogenic exotoxins (SPEs). In addition to the activitieslisted above, some pyrogenic toxins have additional activities that arenot shared by all pyrogenic toxins. For example, the staphylococcalenterotoxins induce emesis and diarrhea when ingested. Structurally, thepyrogenic toxins have varying degrees of relatedness at the amino acidand nucleotide sequence levels. A number of the pyrogenic toxins includea disulfide loop as a structural feature. The staphylococcalenterotoxins have a disulfide loop, as do some others in this family.Examples of other pyrogenic toxins that have a disulfide loop are thestreptococcal superantigen (“SSA”) and streptococcal pyrogenic exotoxinA (“SPEA”).

The pyrogenic toxins have varying degrees of relatedness which providesthe basis for separating some of them informally into subgroups. Onesubgroup includes staphylococcal type B and C enterotoxins (“SEB” and“SEC”), as well as SPEA and SSA. These toxins share between about 49% togreater than 95% amino acid sequence homology (Reda et al, Infect.Immun., 62:1 867-1874: (1994)). Another subgroup of related pyrogenictoxins include staphylococcal type A and E enterotoxins (SEA and SEE)which are 83% homologous to each other (Couch et al, J. Bacteriol.,70:2954-2060 (1988), less so but significantly to SED (Bayles et al., J.Bacteriol., 171:4799-4806 (1989)). The amino acid sequences of thissecond subgroup is more distantly related to SEB, SEC, SPEA, and SSA.Examples of pyrogenic toxins having disulfide bonds are present in bothof these two subgroups. TSST-1 and streptococcal pyrogenic exotoxins Band C (SPEB and SPEC) are examples of a third subgroup of less relatedtoxins. Although toxins from this third subgroup may share someconserved regions (see table 3) with toxins from the other subgroups,there is little overall sequence homology between toxins in the thirdsubgroup and the pyrogenic toxins in the other two subgroups. NeitherTSST-1, SPEB nor SPEC includes a disulfide loop.

The disulfide loop region of a native pyrogenic toxin, such as a nativestaphylococcal enterotoxin, is generally modified through deletion of anumber of amino acid residues within the loop. The modificationtypically includes deletion of amino acid residues within the disulfideloop region and may include one or more substitutions and/or additionsto the remaining loop residues. After modification, the disulfide loopregion typically contains no more than about 10 and, preferably, no morethan about 6 amino acids residues. In another embodiment of theinvention, a modified pyrogenic toxin is formed from a native pyrogenictoxin modified by deletion of at least 40% of the amino acid residueswithin the disulfide loop region, e.g., by deletion of 8 or more aminoacid residues from the disulfide loop region of a native type Cstaphylococcal enterotoxin.

The present invention is also directed to isolated nucleic acids whichinclude a nucleotide sequence encoding a modified pyrogenic toxin.

DETAILED DESCRIPTION OF THE INVENTION

The current invention relates to modified versions of pyrogenic toxins,such as staphylococcal enterotoxins produced by modifications whichinclude deletions in the disulfide bond region. The present inventiondescribes the feasibility of obtaining mutant pyrogenic toxins whichretain biological activity but demonstrate significantly lower toxicityat doses well in excess of the normal lethal dose and levels anticipatedfor human therapeutic use.

The modified pyrogenic toxins are derived from a native pyrogenic toxinhaving a disulfide loop. The terms “disulfide loop” and “disulfide loopregion” are used interchangeably herein. As employed in thisapplication, these terms refer to the sequence of about 10 to about 30amino acid residues forming a loop defined by a disulfide bond in anative pyrogenic toxin. The term “disulfide loop region” also refers tothe corresponding portion of the sequence of a modified pyrogenic toxinwhich has been produced by deletion, substitution or addition of one ormore amino acid residues of the disulfide loop of a native pyrogenictoxin. The disulfide loop region is defined to begin with the N-terminalCys residue and end with the C-terminal Cys residue of the loop, e.g.,amino acid residues 93-110 of staphylococcal enterotoxin C1. As usedherein, the positions of the disulfide loop region for a given nativepyrogenic toxin are numbered beginning with the N-terminal cyteineresidue in the loop, e.g., position 93 of type B or C staphylococcalenterotoxins is also referred to herein as position 1 of the disulfideloop region.

The modification of the disulfide loop typically includes deletion of atleast about 40% of the amino acid residues within the disulfide loop.For example, this generally results in the deletion of at least about 8amino acid residues from the disulfide loop region of an SEC. Examplesof native staphylococcal enterotoxin which can be modified to form thepresent low toxicity toxins include type A, B, C, D, E, G, and Hstaphylococcal enterotoxins. Type C staphylococcal enterotoxins such asstaphylococcal enterotoxin C1, staphylococcal enterotoxin C2,staphylococcal enterotoxin C2, staphylococcal enterotoxin C-MNCopeland,staphylococcal enterotoxin C-4446, staphylococcal enterotoxin C-bovine(GenBank Accession No. L13374), staphylococcal enterotoxin C-canine(GenBank Accession No. V19526) and staphylococcal enterotoxin C-ovine(GenBank Accession No. L13379) are particularly suitable enterotoxinsfor modification by deletion of a portion of the disulfide loop regionto form a staphylococcal enterotoxin with decreased toxicity.

The modified disulfide loop region generally contains no more than about10 amino acid residues and, preferably no more than 6 amino acidresidues. For example, a type C staphylococcal enterotoxin, such asstaphylococcal enterotoxin C1, can be modified to delete amino acidresidues 98-106 (residues 6-14 of the disulfide loop) to form a modifiedstaphylococcal enterotoxin having substantially reduced toxicity. Aneven greater reduction in toxicity is produced by deleting amino acidresidues 95-106 (disulfide loop residues 3-14) of staphylococcalenterotoxin C1. Both of these mutants, despite having substantiallyreduced toxicity, are biologically active as evidenced by their abilityto stimulate the uptake of thymidine by human peripheral bloodmononuclear cells.

In addition to deletion of a number of the disulfide loop residues,substitution of a cysteine residue for the residue at position 2 ofdisulfide loop can contribute to a decrease in the toxicity of apyrogenic toxin such as a type C staphylococcal enterotoxin. In apreferred embodiment, staphylococcal enterotoxin C1 can be modified sothat in addition to deletion of a substantial portion of the residues inthe loop region, the amino acid residue at position 2 of the loop,Tyr-94, is replaced by a cysteine.

Other examples of preferred embodiments of the invention includemodified staphylococcal enterotoxins having no more than 10 amino acidresidues and, preferably, no more than 6 amino acid residues in thedisulfide loop region. Particularly preferred examples includestaphylococcal enterotoxins with no more than about 9 amino acidresidues which include the sequence Cys-Gly-Lys-Thr. One example of sucha mutant is staphylococcal enterotoxin C1 modified to have the disulfideloop region sequence Cys-Cys-Gly-Lys-Thr-Cys.

The methods of this invention employed in preparing mutant enterotoxins,and their screening, analysis, and purification are known in the art anddescribed herein. Site directed mutagenesis can be carried out byinitially cloning the SEC1structural gene SEC_(MNDON) on a 1.4 KbMindITI-BamH1 restriction fragment. A unique SphI restriction site(5′-GCATGC-3′) can be introduced into SEC_(MNDON) in the region codingfor the disulfide loop at nucleotides 301-306 (5′-GTAGGT-3′) using acommercially available kit (Altered Sites in vitro Mutagenesis System,Promega). Potential mutants may be screened by Sph1 digestion andconfirmed by nucleic acid sequence analysis.

Select SEC_(MNDON) deletion mutants were cloned into an E. coli cellline using pMIN164 an E. coli-S. aureus shuttle vector. To facilitateprotein purification, recombinant plasmids from E. coli RR1transformants were transformed into S. aureus RN4220 by standardprotoplast transformation techniques. Plasmids containing cloned toxingenes were maintained in S. aureus RN4220 under erythromycin (50 mg/ml)selection. For purification of native and mutant derivatives of thetoxin recombinants, S. aureus RN4220 cultures were grown in dialyzablebeef heart media supplemented with 1% glucose buffer (330 mM glucose;475 mM NaHCO₃; 680 mM NaCl; 137 mM Na₂HPO₄.H₂O; 28 mM L-glutamine) anderythromycin (50 mg/ml) followed by ethanol precipitation.

Purification of ethanol precipitated proteins was accomplished bypreparative flat bed isoelectric focusing (IEF). Following the IEF run,proteins in select fractions were pooled, dialyzed to remove theamphylotes and then visually assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

The toxicity and biological activity of the mutants was evaluated usingstandard methods known to those skilled in the art as described below.The emetic activity of SEC1, and the SEC1mutant toxins was determined bya modification of the standard monkey feeding assay using young adultpigtail monkeys (M. nemestrina). The animals were manually restrainedwhile the toxin was administered through a nasogastric tube (Infantfeeding tube; Becton Dickinson, Rutherford, N.J.). Toxins were screenedfor retention of emetic activity at a dose of 10 μg/Kg which isapproximately 100 times the minimal emetic dose for SEC1. Non-emetictoxins were tested for residual emetic activity at a high dose of 250μg/kg.

The mitogenic capacities of mutant toxins were compared to that of SEC1native toxin by using human peripheral blood mononuclear cells (PMBC) ina standard 4-day assay. Solutions of native and mutant toxin were addedin triplicate to PBMC cell suspensions, 1×10⁶ cells/ml, in 96-welltissue culture plates. This mixture of cells and toxin was thenincubated at 37° C. under atmospheric conditions of 6% CO₂ for 72 hours.[³H]-thymidine (New Research Products, Boston, Mass.) at a concentrationof 1 μCi/25 μl in a complete RPMI medium was added to each well andallowed to incubate under the same conditions for an additional 18-24hours. After incubation, radiolabeled cellular nucleic acids wereharvested onto glass fiber filters (Skatron, Sterling, Va.) using asemi-automatic cell harvester (Skatron). Lymphocyte proliferation wasquantitated by measuring incorporation of [³H]-thymidine into cellularDNA using a liquid scintillation counter (TRI-CARB 1500 LiquidScintillation Center, Packard, Rockville, Md.).

The ability of a staphylococcal enterotoxin to induce a fever responseand enhance susceptibility of lethal endotoxic shock can be determinedin vivo using a standard rabbit model (Bohach et al., Infect. Immun.,55, 428 (1987)). Following conditioning in a test rack and havingbaseline body temperature recorded, adult New Zealand White rabbits canbe initially intravenously injected with a native or mutant toxin at aconcentration of 10 μg/kg in sterile physiological saline. Sterilesaline and purified SEC1toxin are typically used as negative andpositive controls respectively. Following toxin injection, rabbit bodytemperature is generally monitored rectally every hour for four hours.Four hours after initial treatment, an intravenous injection oflipopolysaccharide (“LPS”) from Salmonella typhimurium (DifcoLaboratories, Detroit, Mich.) is administered intravenously at aconcentration of 10 μg/Kg in sterile saline. Animals are then observedfor signs of shock and mortality for 48 hours after LPS injection.

Cytokine induction may be determined by utilizing isolated mononuclearcells from heparinized venous blood. Briefly, heparinized venous bloodis obtained and layered onto lymphocyte separation medium. The tubes arespun and the mononuclear layer is harvested, washed in PBS, resuspendedin RPMI containing 10% FCS and adjusted to 1×10⁶ cells per ml. Aliquotsof 100 μl are typically placed into 96-well microtiter plates. Theenterotoxins were added in 100 μl to a final concentration of 1 ng/ml.Following incubation at 37° C. for 48-72 hours, the supernatant washarvested can be assayed for cytokines using commercially available kitsfrom R&D Systems Minneapolis, Minn.

The invention will be further described by reference to the followingexamples. These examples illustrate but do not limit the scope of theinvention that has been set forth herein. Variation within the conceptsof the invention will be apparent.

EXAMPLE 1

The SEC1structural gene, SEC_(MNDON), was previously cloned on a 1.4 KbHindIII-BamH1 restriction fragment (Bohach et al., Infect. Immun., 55,428 (1987)). A unique SphI restriction site (5′-GCATGC-3′) wasintroduced into SEC_(MNDON) in the region coding for the disulfide loopat nucleotides 301-306 (5′-GTAGGT-3′) using a commercially available kit(Altered Sites in vitro Mutagenesis System, Promega). Potential mutantswere screened by Sph1 digestion and confirmed by nucleic acid sequenceanalysis.

EXAMPLE 2

The unique Sph1 site was used to linearize the SEC_(MNDON) gene so thatbi-directional deletions could be generated using Bal-31 exonuclease(Boehringer Mannheim, Indianapolis, Ind.). Bal-31 generated deletionmutant plasmids were ligated and transformed into E. coli TG1.Transformants growing on LB-AMP (125 μg/ml) with in-frame stabledeletions were detected by screening with SEC1 specific rabbit antiserain immunodiffusion and analyzed by nucleic acid sequence analysis.Sequencing reactions were done using Sequenase Version 2.0, acommercially available kit (U.S. Biochemical Corp., Cleveland, Ohio).

Three deletion mutants were chosen for detailed analysis based on thesize and location of the deletions in the loop region of the toxin;SEC1-4AA, SCE1-9AA, and SEC1-12AA. Following purification, each of thedisulfide loop mutant toxins could be distinguished from wild typeSEC1on the basis of size when analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

The nucleic acid and amino acid sequences for SEC1and three deletionmutants are shown in Table 4.

EXAMPLE 3 Mutant Toxin Purification

Select SEC_(MNDON) deletion mutants were cloned into E. coli RR1 usingpMIN164, an E. coli-S. aureus shuttle vector. To facilitate proteinpurification, recombinant plasmids from E. coli RR1 transformants weretransformed into S. aureus RN4220 by standard protoplast transformationtechniques. Plasmids containing cloned toxin genes were maintained in S.aureus RN4220 under erythromycin (50 μg/ml) selection. For purificationof the recombinantly produced native and mutant derivatives of thetoxin, S. aureus RN4220 cultures were grown in dialyzable beef heartmedia supplemented with 1% glucose buffer (330 mM glucose; 475 mMNaHCO₃; 680 mM NaCl; 137 mM Na₂HPO₄.H₂O; 28 mM L-glutamine) anderythromycin (50 μg/ml) followed by ethanol precipitation.

Purification of ethanol precipitated proteins was accomplished bypreparative flat bed isoelectric focusing (“IEF”). Following the IEFrun, proteins in select fractions were pooled, dialyzed to remove theampholytes and then visually assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

EXAMPLE 4 Emesis Assay

Assays of the emetic activity of SEC1 and the modified SEC1 toxins wereconducted using a modification of the standard monkey feeding assayusing young adult pigtail monkeys (M. nemestrina). The animals weremanually restrained while the toxin was administered through anasogastric tube (Infant feeding tube; Becton Dickinson, Rutherford,N.J.). Toxins were screened for retention of emetic activity at a doseof 10 μg/Kg which is approximately 100 times the minimal emetic dose forSEC1. Non-emetic toxins were tested for residual emetic activity at ahigh dose of 250 mg/Kg.

It has been previously shown that the minimal emetic dose of wild typeSEC1 for M. nemestrina was 0.1 μg/Kg. For initial experiments in whichthe emetic ability was tested, loop mutant toxins were administered at10 μg/Kg. This insured an excess of toxin over the wild type SEC1minimal emetic dose. Following intragastric toxin inhibition, animalswere observed for at least 12 hours for an emesis response. SEC1-12AAdid not show emesis at the 10 μg/Kg concentration and was subsequentlytested for emesis at a higher toxin concentration, 250 μg/Kg. Even atthis higher doseage level, the SEC1-12AA loop mutant toxin showed noemetic response (See Table 5).

TABLE 5 Emetic Response^(c) of SEC1 Loop Deletion Mutants Dose^(a) SEC1SEC1-4aa SEC1-9aa SEC1-12aa 250 μg/Kg ND^(b) ND ND 0/2  10 μg/Kg 2/2 2/22/2 0/2   1 μg/Kg 2/2 2/2 0/1 ND ^(a)μg Toxin/Kg Body Weight ^(b)ND =Not Determined ^(c)Number animals exhibiting emetic response/Totalnumber of animals

EXAMPLE 5

Pyrogenicity and Enhancement of Lethal Endotoxic Shock

The SEC1 mutant toxin's ability to induce a fever response and enhancesusceptibility of lethal endotoxic shock was determined in vivo using apreviously described rabbit model. Following 1 hour of conditioning in atest rack and having baseline body temperature recorded, adult NewZealand White rabbits were initially intravenously injected with SEC1 orSEC1 mutant toxin at a concentration of 10 μg/Kg in sterilephysiological saline. Sterile saline and purified SEC1 toxin were usedas negative and positive controls, respectively. Following toxininjection, rabbit body temperature was monitored rectally every hour forfour hours. Four hours after initial treatment an intravenous injectionof lipopolysaccharide (LPS) from Salmonella typhimurium (DifcoLaboratories, Detroit, Mich.) was administered intravenously at aconcentration of 10 μg/Kg in sterile saline. Animals were watched forsigns of shock and mortality for 48 hours after LPS injection.

Wild type SEC1, administered at both 10 μg/Kg and 1 μg/Kg, showed atypical temperature rise (Table 6) as well as an enhanced susceptibilityto endotoxic shock (Table 7).

TABLE 6 Maximum Temperature Rise in °C. in Rabbits Given Noted Amount ofNative SEC1, Compound to SEC1-4, SEC1-9, or SEC1-12 Mutant SEC1 SEC1-4aaSEC1-9aa SEC1-12aa  100 μg ND ND ND 0.6    10 μg 1.6   1.46  0.9  0.45   1 μg 1.3   1     0.43 ND  0.1 μg 1.05  0.65  ND ND 0.01 μg 0.4750.425 ND ND

TABLE 7 Lethality in Dutch Belted Rabbits of Native SEC1 and SEC1Deletion Mutants Dose (μg) SEC1 SEC1-4 SEC1-9 SEC1-12 100 ND ND ND 0/310 3/3 3/3 2/3 0/3 1 2/2 3/3 0/3 ND 0.1 3/3 2/3 ND ND 0.01 1/4 0/3 ND ND

SEC1-12AA loop mutant toxin concentration was increased to 100 μg/Kgafter test animals showed both reduced pyrogenic effects and nosusceptibility to endotoxin shock at the initial dose tested, 10 μg/Kg.Following this log fold increase of the SEC-12AA mutant toxin to 100μg/Kg it was seen that the mutant toxin induced a slight temperatureincrease over the initial SEC1-12AA loop mutant toxin concentrationtested but was not lethal in the assay.

EXAMPLE 6 Mitogenicity

The mitogenic capacity of mutant toxin was compared to that of SEC1native toxin by using human peripheral blood mononuclear cells (PMBC) ina standard 4-day assay (Bohach et al., Infect. Immun., 55, 428 (1987)).Human peripheral blood mononuclear cells were isolated from 30 to 60 mlof heparinized blood obtained by venipuncture. The blood was mixed 1:1with Dulbecco's Phosphate Buffered Saline (PBS) (without Calcium ormagnesium) and layered onto Fico/Lite (density 1.079 g/ml, AtlantaBiologicals, Norcross, Ga.) and centrifuged at 400×g for 20 minutes. Theinterface containing the mononuclear cells was removed and washed 3times with PBS. After the last wash, the pellet was resuspended inHank's Balanced Salt solution without calcium or magnesium and layeredon Fetal Bovine Serum (Atlanta Biologicals, Norcross, Ga.) andcentrifuged at 100×g for 10 minutes to remove the platelets. The cellswere washed in Hank's Balanced Salt solution, Cat. No. M1211-021-LV(Atasca, Ill.). A cell count was done by Trypan Blue exclusion.

Solutions of native and mutant toxins were added in triplicate to PMBCcell suspensions, 1×10⁶ cells/ml, in 96 well tissue culture plates. Thismixture of cells and toxin was then incubated at 37° C. underatmospheric conditions at 6% C₀₂ for 72 hours. [³H]-thymidine (NewResearch Products, Boston, Mass.) at a concentration of 1 μCi/25 μl in acomplete RPMI medium was added to each well and allowed to incubateunder the same conditions for an additional 18-24 hours. Afterincubation, radiolabeled cells were harvested onto glass fiber filters(Skatron, Sterling, Va.) using a semi-automatic cell harvester(Skatron). Lymphocyte proliferation was quantitated by measuringincorporation of [³H]-thymidine into cellular DNA using a liquidscintillation counter (TI-CARB 1500 Liquid Scintillation Counter,Packard, Rockville, Md.).

Using SEC1 wild type toxin as a control it was determined that theSEC1-12AA loop mutant toxin showed mitogenic activity at a reducedeffectiveness compared to native SEC1 and the SEC1-4 and SEC1-9 mutantsin stimulating PMBC cells (see Table 8).

TABLE 8 SEC1 Loop Mutant Mitogeneicity [³H]-Thymidine Uptake (in CPM) ofEnterotoxin Stimulated Human Peripheral Blood Mononuclear Cels Dose SEC1SEC1-4AA SEC1-9AA SEC1-12AA  0.1 pg 1918 2067 1067 978    1 pg 8533 74532360 954 0.01 ng 14138 15495 7682 1200  0.1 ng 21557 23689 21481 6500   1 ng 30329 28892 28951 9400 0.01 μg 33089 31640 23333 2700

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains and are hereby incorporated by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

TABLE 1 Amino Acid Sequence of Selected Staphylococcal Enterotoxins                                         40 *   *    *  *    *  *    *                *?                *   *  * * * SEC1 1ESQPDPTPDELHKASKFTGLMENMKVLYDDHYVSATKVKSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEGLAKKYKDE80 (SEQ. ID. NO:1) SEC2 1ESQPDPTPDELHKSSEFTGTMGNMKYLYDDHYVSATKVMSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDE80 (SEQ. ID. NO:2) SEC3-FR1913 1ESQPDPMPDDLHKSSEFTGTMGNMKYLYDDHYVSATKVKSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDE80 (SEQ. ID. NO:3) SEC3-FR1909 1ESQPDPMPDDLHKSSEFTGTMGNMKYLYDDHYVSATKVKSVDKFLAHDLIYNINDKKLNNYDKVKTELLNEDLANKYKDE80 (SEQ. ID. NO:4) SEC-MNCopeland 1ESQPDPTPDELHKSSEFTGTMGNMKYLYDDHYVSATKVKSVDKFLAHDLIYNINDKKLKNYDKVKTELLNEDLAKKYKDE80 (SEQ. ID. NO:5) SEC-4446 1ESQPDPMPDDLHKSSEFTGTMGNMKYLYDDHYVSATKVKSVDKFLAHDLIYNISDKRLKNYDKVKTELLNEDLAKKYKDE80 (SEQ. ID. NO:6) SEC-bovine 1ESQPDPTPDELHKASKFTGLMENMKVLYDDRYVSATKVKSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDE80 (SEQ. ID. NO:7) SEC-ovine 1ESQPDPTPDELHKASKFTGLMENMKVLYDDRYVSATKVKSVDKFLAHDLIYNISDKKLKNYDKVKTELLNEDLAKKYKDE80 (SEQ. ID. NO:8)                                   120   *            *            ?       *     * SEC1 81VVDVYGSNYYVNCYFSSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLIRVYENKRNTISFEVQTDKKSVTAQELD160 SEC2 81VVDVYGSNYYVNCYFSSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLIRVYENKRNTISFEVQTDKKSVTAQELD160 SEC3-FR1913 81VVDVYGSNYYVNCYFSSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLYRVYENKRNTISFEVQTDKKSVTAQELD160 SEC3-FR1909 81VVDVYGSNYYVNCYFSSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLIRVYENKRNTISFEVQTDKKSVTAQELD160 SEC-MNCopeland 81VVDVYGSNYYVNCYFSSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLIRVYENKRNTISFEVQTDKKSVTAQELD160 SEC-4446 81VVDVYGSNYYVNCYFSSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLVRVYENKRNTISFEVQTDKKSVTAQELD160 SEC-bovine 81VVDVYGSNYYVNCYFFSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLIRVYENKRNTISFEVQTDKKSVTAQELD160 SEC-ovine 81VVDVYGSNYYVNCCFFSKDNVGKVTGGKTCMYGGITKHEGNHFDNGNLQNVLIRVYENKRNTISFEVQTDKKSVTAQELD160                               200   *                     *           ?              *  *   *    * SEC1 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFWYDMMPAPGDKFDQSKYLMMYNDNKTVDSKSVKIEVHLTTKNGX240 SEC2 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFQYDMMPAPGDKFDQSKVLMMYNDNKTVDSKSVKIEVHLTTKNGX240 SEC3-FR1913 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFQYDMMPAPGDKFDQSKYLMMYNDNKTVDSKSVKIEVHLTTKNGX240 SEC3-FR1909 161IKARNFLINKKNLYEFNSSPYETGYIKFIESNGNTFWYDMMPAPGDKFDQSKYLMIYKDNKMVDSKSVKIEVHLTTKNGX240 SEC-MNCopeland 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFQYDMMPAPGDKFDQSKYLMMYNDNKTVDSKRVKIEVHLTTKNGX240 SEC-4446 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFQYDMMPAPGDKFDQSKYLMMYNDNKTVDSKRVKIEVHLTTKNGX240 SEC-bovine 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFQYDMMPAPGDKFDQSKYLMMYNDNKTVDSKRVKIEVHLTTKNGX240 SEC-ovine 161IKARNFLINKKNLYEFNSSPYETGYIKFIENNGNTFQYDMMPAPGDKFDQSKYLMMYNDNKTVDSKRVKIEVHLTTKNGX240

TABLE 3A Conserved Regions of Enterotoxin Molecules* Toxin Residue #Region 1 SEA  79 K Y K G K K V D L Y G (SEQ. ID. NO:18) SEB  76 K Y K DK Y V D V F G (SEQ. ID. NO:19) SEC1  76 K Y K D E V V D V Y G (SEQ. ID.NO:20) SEC2  76 K Y K D E V V D V Y G (SEQ. ID. NO:21) SEC3  76 K Y K DE V V D V Y G (SEQ. ID. NO:22) SED  74 H F K S K N V D V Y P (SEQ. ID.NO:23) SEE  76 K Y K G K K V D L Y G (SEQ. ID. NO:24) SPEA  70 L F K D KN V D I Y G (SEQ. ID. NO:25) SPEC  63 F K R D D H V D V F G (SEQ. ID.NO:26) TSST-1  56 F T K G E K V D L N T (SEQ. ID. NO:27) Region 3 SEA147 K K N V T V Q E L D L Q A R R Y L (SEQ. ID. NO:28) SEB 152 K K K V TA Q E L D Y L T R H Y L (SEQ. ID. NO:29) SEC1 151 K K S V T A Q E L D IK A R N F L (SEQ. ID. NO:30) SEC2 151 K K S V T A Q E L D I K A R N F L(SEQ. ID. NO:31) SEC3 151 K K S V T A Q E L D I K A R N F L (SEQ. ID.NO:32) SED 142 K K N V T V Q E L D A Q A R R Y L (SEQ. ID. NO:33) SEE144 K K E V T V Q E L D L Q A R H Y L (SEQ. ID. NO:34) SPEA 137 K K M VT A Q E L D Y K V R K Y L (SEQ. ID. NO:35) SPEC 124 K D I V T F Q E I DF K I R K Y L (SEQ. ID. NO:36) TSST-1 121 K K - - - - Q - L - I (SEQ.ID. NO:37) TSST-1 129           L D F E I R H Q L (SEQ. ID. NO:38) *FromHoffmann et al., Infect Immunol 62:3396 (1994).

TABLE 3B Conserved Regions of Enterotoxin Molecules (Cont.)* ToxinResidue # Region 2 SEA 106 C M Y G G V I L H D N N (SEQ. ID. NO:39) SEB113 C M Y G G V T E H N G N (SEQ. ID. NO:40) SEC1 110 C M Y G G I T K HE G N (SEQ. ID. NO:41) SEC2 110 C M Y G G I T K H E G N (SEQ. ID. NO:42)SEC3 110 C M Y G G I T K H E G N (SEQ. ID. NO:43) SED 101 C T Y G G V TP H E G N (SEQ. ID. NO:44) SEE 103 C M Y G G V T L H D N N (SEQ. ID.NO:45) SPEA  98 C I Y G G V T N H E G N (SEQ. ID. NO:46) SPEC  85 Y I YG G I T P A Q N N (SEQ. ID. NO:47) TSST-1  83 F Q I S G V T N T E K L(SEQ. ID. NO:48) Region 4 SEA 209 L L R I Y R D N K T I N S E (SEQ. ID.NO:49) SEB 213 Y L M M V N D N K M V D S K (SEQ. ID. NO:50) SEC1 213 Y LM M Y N D N K T V D S K (SEQ. ID. NO:51) SEC2 213 Y L M M Y N D N K T VD S K (SEQ. ID. NO:52) SEC3 213 Y L M I Y K D N K M V D S K (SEQ. ID.NO:53) SED 204 Q L R I Y S D N K T L S T E (SEQ. ID. NO:54) SEE 206 L LR I Y R D N K T I N S E (SEQ. ID. NO:55) SPEA 197 Y L M I Y K D M E T LD S N (SEQ. ID. NO:56) SPEC 184 I F A K Y K D N R I I N M K (SEQ. ID.NO:57) TSST-1 179 P P I N I D E I K T I E A E (SEQ. ID. NO:58) *FromHoffmann et al., Infect Immunol 62:3396 (1994).

TABLE 4 SEC1 LOOP MUTANTS AMINO ACID # 93  94  95  96  97  98  99  100101 102 103 104 105 106 107 108 109 110 SEC1 (wild type) AMINO ACID CysTyr Phe Ser Ser Lys Asp Asn Val Gly Lys Val Thr Gly Gly Lys Thr Cys(SEQ. ID. NO:60) NUCLElC ACID TGC TAT TTT TCA TCC AAA GAT AAT GTA GGTAAA GTT ACA GGT GGC AAA ACT TGT (SEQ. ID. NO:59) SEC1 Loop                        301 -   306 Deletion Mutants 4 A.A. MUTANT CysTyr Phe Ser Ser Lys Asp Asn Ala                 Gly Gly Lys Thr Cys(SEQ. ID. NO:62) TGC TAT TTT TCA TCC AAA GAT AAT GCA -------         GGTGGC AAA ACT TGT (SEQ. ID. NO:61) 9 A.A. MUTANT Cys Tyr Phe SerSer                                     Gly Lys Thr Cys (SEQ. ID. NO:64)TGC TAT TTT TCA TCC ---------------                     GGC AAA ACT TGT(SEQ. ID. NO:63) 12 A.A MUTANT CysCys                                                 Gly Lys Thr Cys(SEQ. ID. NO:66) TGCT-- --------------------                        GT  GGC AAA ACT TGT(SEQ. ID. NO:65)

1. A toxin, comprising a polypeptide sequence selected from the group,consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; wherein thepolypeptide sequence is mutated in the region from residue 93 to residue110 to contain no more than 10 amino acid residues; wherein toxicity isreduced in comparison to the unmutated native toxin.
 2. The toxin ofclaim 1 wherein the polypeptide sequence is mutated in the region fromresidue 93 to residue 110 to contain no more than 6 amino acid residues.3. The toxin of claim 1, wherein the polypeptide sequence comprises SEQID NO: 1 and the mutation comprises deletion of amino acid residues95-106 of SEQ ID NO:
 1. 4. The toxin of claim 1, wherein the polypeptidesequence comprises SEQ ID NO: 1 and the mutation further comprises acysteine residue at position 94 of SEQ ID NO:
 1. 5. The toxin of claim 1having an emetic response inducing activity decreased by at least100-fold in comparison to the native toxin.
 6. The toxin of claim 1having a fever inducing activity decreased by at least 100-fold incomparison to the native toxin.
 7. The toxin of claim 1 having an LD50in Dutch Belted rabbits which is at least 100-fold higher than thenative toxin.
 8. A staphylococcal toxin, comprising an amino acidsequence of a Type C staphylococcal toxin having a disulfide loop regionwherein at least 40% of the amino acid residues within the disulfideloop region are deleted and wherein toxicity is reduced in comparison tothe unmutated native toxin.
 9. The staphylococcal toxin of claim 8comprising a cysteine residue at position 2 of the disulfide loopregion, wherein the disulfide loop region, before deletion of at least40% of the amino acid residues, is selected from the group consisting ofSEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:
 17. 10. Astaphylococcal toxin, wherein the staphylococcal toxin comprises adisulfide loop region comprising SEQ ID NO: 66(Cys-Cys-Gly-Lys-Thr-Cys)and wherein toxicity is reduced in comparisonto the unmutated native toxin.
 11. The toxin of claim 1, wherein thepolypeptide sequence comprises SEQ ID NO: 1 and the mutation comprises adeletion of amino acid residues 98-106 of SEQ ID NO:
 1. 12. The toxin ofclaim 11 wherein the mutation comprises deletion of at least 12 aminoacid residues in the region from residue 93 to residue 110.